OBJECTIVE: To determine the degree of blood cell contamination in GC preparations; to assess techniques of GC purification; and to determine possible effects of contaminating cells and purification techniques on cultured GC in terms of steroid hormone production. DESIGN: Contamination of GC by white blood cells was assessed by Wright's stain and immunohistochemistry. Purification was attempted by: (1) Ficoll density centrifugation (to remove polymorphonuclear leukocytes [PML]); (2) incubation in tissue culture plastic dishes (to remove adherent monocyte/macrophages); and (3) incubation in the presence of high salt (to remove lymphocytes). RESULTS: Ficoll density centrifugation reduced PML to 2% of total cells, incubation in plastic dishes reduced monocyte/macrophage contamination from 6% to 7% down to less than 1%, and high-salt incubation reduced lymphocyte contamination from 10% to 12% down to 4%. Granulosa cells plated after preparation with addition of high salt showed increased progesterone production, which could not be entirely explained by the removal of contaminating lymphocytes. CONCLUSION: These results indicate that the human GC culture system is more complex than is often assumed, and methods that remove a majority of these white blood cells are presented.
OBJECTIVE: To determine the degree of blood cell contamination in GC preparations; to assess techniques of GC purification; and to determine possible effects of contaminating cells and purification techniques on cultured GC in terms of steroid hormone production. DESIGN: Contamination of GC by white blood cells was assessed by Wright's stain and immunohistochemistry. Purification was attempted by: (1) Ficoll density centrifugation (to remove polymorphonuclear leukocytes [PML]); (2) incubation in tissue culture plastic dishes (to remove adherent monocyte/macrophages); and (3) incubation in the presence of high salt (to remove lymphocytes). RESULTS: Ficoll density centrifugation reduced PML to 2% of total cells, incubation in plastic dishes reduced monocyte/macrophage contamination from 6% to 7% down to less than 1%, and high-salt incubation reduced lymphocyte contamination from 10% to 12% down to 4%. Granulosa cells plated after preparation with addition of high salt showed increased progesterone production, which could not be entirely explained by the removal of contaminating lymphocytes. CONCLUSION: These results indicate that the human GC culture system is more complex than is often assumed, and methods that remove a majority of these white blood cells are presented.
Authors: Edo Dzafic; Martin Stimpfel; Srdjan Novakovic; Petra Cerkovnik; Irma Virant-Klun Journal: Biomed Res Int Date: 2014-03-03 Impact factor: 3.411